2-amino-4 5 6 7-tetrahydro - 4 7 - ethanothieno(2 3-b)pyridines an pharmaceutically acceptable salts thereof

ABSTRACT

THIS INVENTION RELATES TO THE DISCOVERY OF A GROUP OF COMPOUNDS HEREIN DESCRIBED AS 2-AMINO-4,5,6,7-TETRAHYDRO-4,7-ETHANOTHIENO(2,3,B)PYRIDINES WHICH ARE EFFECTIVE TOPICALLY AGAINST HERPES SIMPLEX/H4/VIRUS, HERPES SIMPLEX/2/ALA/SM/VIRUS AND ADENOVIRUS-2.

United States Patent 3,644,379 2-AMINO-4,5,6,7-TE'IRAHYDRO 4,7 ETHANO-THIENO[2,3-b]PYRIDINES AND PHARMACEU- TICALLY ACCEPTABLE SALTS THEREOFIan Wellings, Wilmington, Del., assignor to E. I. du Pont de Nemours andCompany, Wilmington, Del. No Drawing. Filed Oct. 23, 1969, Ser. No.868,898 Int. Cl. C07d 31/50 U.S. Cl. 260294.8 B 7 Claims ABSTRACT OF THEDISCLOSURE This invention relates to the discovery of a group ofcompounds herein described as2-amino-4,5,6,7-tetrahydro-4,7-ethanothieno[2,3-b]pyridines which areeffective topically against Herpes simplex/H /virus, Herpes simplex/ 2/ALA/SM virus and adenovirus-2.

SUMMARY OF THE INVENTION This invention relates to compounds of theformula Lt 5(1 R1 6 l 2 N s: 2 7 l where R, is

where R is hydrogen, alkyl of one through six carbon atoms;

X is oxygen or sulfur;

R is hydrogen or alkyl of one through four carbon atoms;

R, is hydrogen or alkyl of one through four carbon atoms and thepharmaceutically acceptable non-toxic salts of said compounds.

Another aspect of the invention is to demonstrate the inhibition ofgrowth of Herpes simplex/H /virus and Herpes simplex/Z/ALA/SM virus whenat least 10 mcg./ ml. are applied to a culture medium containinginfected rabbit kidney cells or mouse embryo cells respectively.

In addition, the antiviral activity of the compounds of this inventioncan be further demonstrated against adenovirus-2 when an agar mediumcontaining HEp-Z-cell monolayers and 80 mcg. of the compound,2-arnino-4,5,6,7-tetrahydro-4,7-ethanothieno/2,3-b/pyridine 3carbonitrile shows no damage to said HEp-2-cells after an incubationperiod of 7 days at 37 C. as compared to controls.

Still another aspect of the invention is to show that shaved guinea pigsinfected intradermally with Herpes simplex/H /virus showed only 38%positive lesions after 7 days as compared to control animals whichshowed 86% positive lesions when a compound of the invention wasadministed topically.

DESCRIPTION OF THE INVENTION As summarized above, this invention relatesto compounds of the formula:

"ice

where R is X B3 CN, -COOR2 or -ii-N R4 where R is hydrogen or alkyl ofone through six carbon atoms; X is oxygen or sulfur; R is hydrogen oralkyl of one through four carbon atoms; R, is hydrogen or alkyl of onethrough four carbon atoms;

and the pharmaceutically acceptable non-toxic salts of said compounds.2-arnino-4,5,6,7-tetrahydro-4,7-ethanothieno [2,3-b]

pyridine-3-carbonitrile.2-amino-4,5,6,7-tetrahydro-4,7-ethanothieno[2,3-b]

pyridine-3-carboxylic acid ethylester. 2-amino-4,5,6,7-tetrahydro-4,7-ethanothieno [2,3 -b] pyridine-3-carboxamide.

It will be understood that the term non-toxic pharmaceuticallyacceptable salts includes those salts of the compounds of this inventionwhich are suitable for administration to warm-blooded animals.Representative of such salts are the hydrochlorides, hydrobromides,sulfate, phosphates, acetates, nitrates, succinates, adipates,propionates, tartrates, cyclohexylsulfamates, citrates, bicarbonates,and pamoates. Of these the hydrochloride salt is preferred.

Illustrative of the compounds of this invention are the following:2-amino-4,5,6,7-tetrahydro-4,7-ethanothieno[2,3-b]

pyridine-3-carbonitrile. methyl2-amino-4,5,6,7-tetrahydro-4,7-ethanothieno[2,3-b]pyridine-3-carboxylate. ethyl2-amino-4,5,6,7-tetrahydro-4,7-ethanothieno[2,3-b]pyridine-3-carboxylate. isobutyl2-amino-4,5,6,7-tetrahydro-4,7-ethanothieno[2,3-b]pyridine-3-carboxylate.2-amino-4,5,6,7-tetrahydro-4,7-ethanothieno[2,3-b]

pyridine-3-carboxylic acid. 2-amino-4,5,6,7-tetrahydro-4,7-ethanothieno[2,3-b]

pyridine-3-carboxamide.N-methyl-2-amino-4,5,6,7-tetrahydro-4,7-ethanothieno[2,3-b]pyridine-3-carboxamide.N-isopropyl-2-amino-4,5,6,7-tetrahydro-4,7-ethanothieno [2,3-b]pyridine-3-carboxamide. N,N-dimethyl-2-amino-4,5,6,7-tetrahydro-4,7-

ethanothieno[2,3-b]pyridine-3-carboxamide.N-methyl-N-butyl-Z-amino-4,5,6,7-tetrahydro-4,7-

ethanothieno[2,3-b]pyridine-3-carboxamide.2-amino-4,5,6,7-tetrahydro-4,7-ethanothieno[2,3-b]

pyridine-3-thiocarboxamide.N-methyl-2-amino-4,5,6,7-tetrahydro-4,7-ethanothieno[2,3-b]pyridine-3-thiocarboxamide.N-methyl-N-propyl-2-amino-4,5,6,7-tetrahydro-4,7-

ethanothieno[2,3-b]pyridine-3-thiocarboxamide.

The compounds of this invention are prepared by treating3-quinuclidinone with a mixture of an appropriate nitrile, sulfur andmorpholine.

The following examples are presented to further illustrate the method ofpreparing the compounds of this invention.

Example 1.2-amino-4,5,6,7-tetrahydro-4,7-ethanothieno[2,3-b]pyridine-3-carbonitrile A solution of 0.1 mole of malonitrile in20 ml. of ethanol is added to a stirred mixture of 0.1 mole of 3-quinuclidinone, 0.11 mole of sulfur and 3.0 ml. of morpholine in ml. ofethanol. The reaction mixture is then warmed to 50 C. and held at thattemperature for 4 hours under an atmosphere of nitrogen. After coolingto ice bath temperature, the precipitated solids are removed byfiltration and recrystallized from aqueous dimethylformamide to give2-amino-4,5,6,7-tetrahydro-4,7-ethanothieno[2,3-b]pyridine-3-carbonitrile, M.P. 200-201 C.

The hydrochloride salt of this compound is prepared by passing dryhydrogen chloride gas into an ethanolic solution of the base. The saltis recrystallized from ethanol/ water to give2-amino-4,5,6,7-tetrahydro-4,7-ethanothieno[2,3-b]pyridine-3-carbonitrile hydrochloride, M.P. 273- 274 C.

Example 2.Ethyl 2-amino-4,5,6,7-tetrahydro-4,7- ethanothieno [2,3-b]pyridine-3-carboxylate A solution of 0.1 mole of ethyl cyanoacetate in20 ml. of ethanol is added to a stirred mixture of 0.1 mole of 3-quinuclidinone, 0.11 mole of sulfur and 3.0 ml. of morpholine in asolution of 80 ml. of ethanol. The reaction mixture is then warmed to 40C. and stirred overnight at this temperature. After cooling to roomtemperature, the mixture is filtered and the filtrate is poured into 400ml. of water containing 4 ml. of glacial acetic acid to give a semisolidmass. The aqueous phase is decanted ofr" and the semisolid material iswashed four times with 200 ml. of water containing 2 ml. of glacialacetic acid. The residue is recrystallized from acetonitrile to giveethyl 2- amino 4,5,6,7 tetrahydro4,7-ethanothieno[2,3-b1pyridine-3-carboxylate, M.P. 148-149 C.

The hydrochloride salt of this compound is prepared by passing dryhydrogen chloride gas into an ethanolic solution of the base. The saltis recrystallized from ethanol/water to give ethyl2-amino-4,5,6,7-tetrahydro- 4,7-ethanothieno[2,3-b1pyridine 3carboxylate hydrochloride, M.P. 259-260 C.

Examples 3-5 The procedure of Example 2 is repeated, substituting theindicated cyanoacetic ester for the ethyl cyanoacetate of Example 2 toobtain the indicated product.

Example6.-2-amino-4,5,6,7-tetrahydro-4,7-ethanothieno[2,3-b]pyridine-3-carboxylicacid A solution of 0.1 mole of ethyl2-amino-4,5,6,7-tetrahydro-4,7-ethanothieno[2,3-b] pyridine 3carboxylate in 100 ml. of acetic anhydride is warmed to 50 C. for 2hours. The reaction mixture is then cooled to room temperature andpoured on a mixture of 200 gm. of ice and 200 ml. of water and stirredfor one hour. The mixture is neutralized by adding concentrated ammoniumhydroxide while keeping the temperature below 20 C. The precipitatedsolid is recrystallized from acetonitrile to give ethyl Z-acetamido4,5,6,7 tetrahydro-4,7-ethanothieno[2,3-b]pyridine-3-carboxylate, M.P.194-195 C.

A solution of 0.1 mole of this ester and 12.0 gm. of sodium hydroxide in175 m1. of water and 125 ml. of dioxane is refluxed for 1 hours. Thereaction mixture is concentrated in vacuo to about one half of itsoriginal volume and any insoluble material is removed by filtration. Thefiltrate is stirred and cooled to C. and glacial acetic acid is addedslowly until the solution reaches pH 7. The precipitate which forms isfiltered oil and dried in a desiccator over calcium chloride to yield2-amino-4,5,6,7- tetrahydro 4,7 ethanothieno[2,3-b]pyridine-3-carboxylicacid.

4 Example 7.--2-amino-4,5,6,7-tetrahydro-4,7-ethanothieno[2,3-b]pyridine-3-carboxamide A solution of 0.1 mole of 2-cyanoacetamide in 50ml. of dimethylformamide is added to a stirred mixture of 0.1 mole of3-quinuclidinone, 0.11 mole of sulfur and 3.0 ml. of morpholine in 50ml. of dimethylformamide. The reaction mixture is warmed to 50 C. andheld at that temperature for 4 hours under an atmosphere of nitrogen.After cooling to room temperature, the mixture is poured into 500 ml. ofwater containing 5 ml. of glacial acetic acid to give a precipitatewhich is collected by filtration. Crystallization. of this solid fromacetonitrile gives 2- amino 4,5,6,7 tetrahydro 4,7 ethanothieno[2,3-b]pyridine-3-carboxamide, M.P. -197 C.

Examples 8-10 The procedure of Example 7 is repeated, substituting theindicated cyanoacetamide for the 2-cycanoacetamide of Example 7 toobtain the indicated product.

Ex. Cyanoaeetamide Product 8 N,N-dimethyl-2-N,N-dirnethyl2-amiu0-4,5,6,7-t etrahycyanoacetamide.dro-4,7-ethanothieno[2,3,b]pyridlne-3- carb oxamide. 9.N-propyl-2'cyano- N-propyl-2-amino-4,5,6,7-tetrahydro-4,7-

acetamide. ethanothieno-[2,3-b]pyridine-S-carboxamlde.

N-ethyl-N-methyl-2-amino-4,5,6,7-tetrahydro-4,7-ethanothieno[2,3-b1pyridine-3-earboxamide.

10. N-ethyl-N-methyl-2- cyanoacetamide.

Example 1 1 .2-amino-4,5 ,6,7-tetrahydro-4,7-ethanothieno [2,3-b]pyridine-B-tlriocarboxamide A solution of 0.1 mole of2-amino-4,5,6,7-tetrahydro-4,7-ethanothieno[2,3-b]pyridine-3-carbonitrile in 30 ml. of pyridine and0.1 mole of triethylamine is treated with dry hydrogen sulfide bypassing the gas through the solution in a steady stream for 2-3 hours.The mixture is then poured into water and the precipitate which forms isfiltered off and dried to give 2-amino-4,5,6,7-tetrahydro-4,7-ethanothieno[2,3-b]pyridine-3-thiocarboxamide.

Example 12.N-propyl-2-amino-4,5,6,7-tetrahydro-4,7- ethanothieno [2,3-b]pyridine-3 -thiocarboxamide Example13.-N,N-dimethyl-2-amino-4,5,6,7-tetrahydro- 4,7-ethanothieno [2,3-b]pyridine-3-thiocarb oxamide A mixture of 0.1 mole ofN,N-dimethyl-2-amino- 4,5,6,7 tetrahydro 4,7 ethanothieno[2,3-b1pyridine-3- carboxamide and 20.0 gm. of phosphorus pentasulfidein 250 ml. of xylene is stirred vigorously and refluxed for 2 hours. Themixture is then cooled in an ice-bath and an excess of 10% sodiumhydroxide solution is added dropwise. The mixture is stirred for anadditional 15 minutes and then filtered to give a residue ofN,N-dimethyl-2- amino 4,5,6,7tetrahydro-4,7-ethanothieno[2,3-b1pyridine-3-thiocarboxamide.

The active ingredient of this invention can be employed in usefulcompositions according to the present invention in such dosage forms astablets, capsules, powder, packets, or liquid solutions, suspensions, orelixirs, for oral administration or liquid for parenteral use, and incertain cases, suspensions for parenteral use (except intraveous). Thecompounds may also be applied topically in solution, lotion or ointmentform. In the pharmaceutical compositions of this invention the activeingredient will ordinarily always be present in an amount of at least0.5% by weight based on the total weight of the composition and not morethan 99% by weight.

Besides the active ingredient of this invention, the antiviralcomposition will contain a solid or liquid nontoxic pharmace'uticalcarrier for the active ingredient.

In one embodiment of a pharmaceutical composition of this invention, thesolid carrier is a capsule which can be of the ordinary gelatin type. Inthe capsule will be from about 3099% by weight of a compound of Formula1 and 701% of a carrier. In another embodiment, the active ingredient istableted with or without adjuvants. In yet another embodiment, theactive ingredient is put into powder packets and employed. In thesecapsules, tablets and powders the active ingredient will generallyconstitute from about 5% to about 99% and preferably from 25% to 90% byweight. These dosage forms preferably contain from about 5 to about 500milligrams of active ingredient, with from about 25 to about 250 mostpreferred.

The pharmaceutical carrier can, as previously indicated, be asterileliquid such as water and oils, including those of petroleum,animal, vegetable or synthetic origin, for example peanut oil, soybeanoil, mineral oil, sesame oil, and the like. In general, water saline,aqueous dextrose (glucose) and related sugar solutions and glycols suchas propylene glycol or polyethylene glycols are preferred liquidcarriers, particularly for injectable solutions. Sterile injectablesolutions such as saline will ordinarily contain from about 0.5% to 25and preferably about 1 to by Weight of the active ingredient.

As mentioned above, oral administration can be in a suitable suspensionor syrup, in which the active ingredient ordinarily will constitute fromabout 0.5 to 10% and preferably about 2 to 5%, by weight. Thepharmaceutical carrier in such composition can be a watery vehicle suchas an aromatic water, a syrup or a pharmaceutical mucilage.

Suitable pharmaceutical carriers are described in RemingtonsPharmaceutical Sciences by E. W. Martin, a well-known reference text inthis field.

In addition to the exemplary illustrations above, the following examplesfurther explain the present invention.

Example 14 A large number of unit capsules are prepared by fillingstandard two-piece hard gelatin capsules weighing about 50 milligramseach with 250 milligrams of powdered 2 amino 4,5,6,7 tetrahydro 4,7ethanothieno- [2,3-b] pyridine 3 -carbonitrile, hydrochloride, 110milligrams of lactose, 1 milligram of Cab-o-Sil, 8 milligrains ofmagnesium stearate and 32 milligrams of talc.

Example 15 Soft gelatin capsules can be prepared by dissolving orsuspending powdered 2-amino 4,5,6,7 tetrahydro- 4,7 ethanothieno[2,3-b]pyridine 3 carbonitrile in soybean oil.

Example 16 A large number of tablets are prepared by conventionalprocedures so that the dosage unit is 50' milligrams of activeingredient, 5 milligrams of gelatin, 1.5 milligrams of magnesiumstearate and 100 milligrams of lactose. Slow release pills or tabletscan also be prepared by applying appropriate coatings. A sugar coatingmay be applied to increase palatability.

Example 17 A parenteral composition suitable for administration byinjection is prepared by stirring 5% by weight of ethyl 2 amino 4,5,6,7tetrahydro 4,7 ethanothieno- [2,3-b] pyridine 3 carboxylatehydrochloride in sodium chloride injection, and filtering the solutionthrough a sterilizing filter.

Example 18 A composition for topical application is prepared bytriturating 10% by weight of the active ingredient in hydrophilicointment, U.S.P.

A large variety of compositions according to this invention can thusreadily be made by substituting other compounds of this invention, andincluding specifically but not limited to compounds of this inventionthat have specifically been named hereinbefore. The compounds will beused in the amounts indicated in accordance with procedures well knownand described in the Martin text mentioned above.

It will be understood that a warm-blooded animal is a member of theanimal kingdom possessed of a homeostatic mechanism and includes mammalsand birds.

The antiviral effectiveness of the compounds of this invention can bedemonstrated against Herpes virus when a medium containing at least 10mcg./ml. of Z-amino- 4,5,6,7 tetrahydro 4,7ethanothieno[2,3-b1pyridine-3- carbonitrile, hydrochloride is applied torabbit kidney cells which have been infected with 200 plaque formingunits of Herpes simplex/H4/virus with the result that completeinhibition of the cytopathic effect of the virus is noted.

2-amino 4,5,6,7 tetrahydro 4,7 ethanothieno[2,3- b]pyridine 3carbonitrile hydrochloride when applied at a concentration of tenmeg/ml. is also effective in preventing damage to mouse embryo cellswhich have been infected with 300 plaque forming units of Herpes sim plex/ 2./ ALA/ SM virus.

A local application of 20 mcg. of 2-amino 4,5,6,7- tetrahydro 4,7ethanothieno[2,3-b1pyridine 3 carboxylic acid, ethyl ester to a cellmonolayer of rabbit kidney cells infected with Herpes simplex/H4 viruseffectively inhibits the cytopathic effect of the virus on said cells.

A local application of '80 mcg. of 2 amino 4,5,6,7- tetrahydro 4,7ethanothieno[2,3-b1pyridine 3 carboxamide to a cell monolayer of rabbitkidney cells infected with Herpes simplex/H4 virus effectively inhibitsthe cytopathic effect of the virus on said cells.

The effectiveness of 2 amino 4,5,6,7, tetrahydro- 4,7ethanothieno[2,3-b]pyridine 3 carbonitrile, hydrochloride in combatingHerpes simplex/H4 infections in guinea pigs can be demonstrated asfollows:

Guinea pigs are shaved and infected intradermally on 18 sites, 9 sitesper side, with Herpes simplex/H4 virus. The test compound, 2 amino4,5,6,7 tetrahydro-4,7- ethanothieno[2,3-b]pyridine 3 carbonitrile,dissolved in a vehicle consisting of 90% dimethylsulfoxide and 10% wateris administered topically at 24, 48, 72 and 96 hours after infection.Seven days after infection the guinea pigs treated with the abovemixture are compared to a control group of guinea pigs infected withsaid virus but which are treated with the vehicle only. Control animalsshow 86% positive lesions whereas animals treated with 0.375 mg./ siteof 2 amino 4,5,6,7- tetrahydro 4,7 ethanothieno[2,3ab]pyridine 3carbonitrile hydrochloride show only 38% positive lesions.

The antiviral effectiveness of the compounds of this invention can bedemonstrated against adenovirus when a medium containing mcg. of 2 amino4,5,6,7 tetrahydro 4,7 ethanothieno[2,3-b] pyridine 3 carbonitrile islocally applied to an agar medium containing HE -2 cell monolayers whichare infected with Adenovirus 2 and incubated at 37 C. for seven days.After the incubation period, it is found that the tissue surrounding themedicated area is normal While other areas of the infected tissue isheavily damaged. The result demonstrates that the test compoundeffectively inhibits the growth of adenovirus-2.

7 I claim: 1. A compound of the formula:

(f iit where R is where R is hydrogen or alkyl of one through six carbonatoms; X is oxygen or sulfur; R is hydrogen or alkyl of one through fourcarbon atoms; R; is hydrogen or alkyl of one through four carbon atoms;and the pharmaceutically acceptable non-toxic salts of said compounds.

2. The compound of claim 1 which is 2-amino-4,5,6,7-

8 tetrahydro-4,7-ethanothien0[2,3-b1pyridine 3 carbonitrile.

3. A pharmaceutically acceptable non-toxic salt of the compound of claim2.

4. The compound of claim 1 which is 2-amino-4,5,6,7- tetrahydro 4,7ethanothieno[2,3-b]pyridine-B-carboxylic acid ethyl ester.

5. A pharmaceutically acceptable non-toxic salt of the compound of claim4.

6. The compound of claim 1 which is 2-amino-4,5,6,7tetrahydro-4,7-ethanothieno[2,3-b] pyridine 3 carboxamide.

7. A pharmaceutically acceptable non-toxic salt of the compound of claim6.

References Cited Antibiotic News, vol. 5, No. 9, October 1968, pp. 1 and3.

ALAN L. ROTMAN, Primary Examiner US. Cl. X.R. 424263, 266

